During the coming year we will extend our studies on the various phenotypes of mutations in the pyrA gene of Salmonella typhimurium, the gene that encodes carbamylphosphate synthetase. Having established that the arginine-sensitive phenotype is a consequence of inability of the two unequal subunits to assemble and form the holoenzyme in the absence of ornithine transcarbamylase (owing to the repression of the latter enzyme by arginine) we will test the generality of this type of defect by studying the uracil-sensitive and arginine- or uracil-sensitive phenotypes. Our working hypothesis attributes these phenotypes to the requirement for sub-unit assembly to the presence of aspartate transcarbamylase and aspartate transcarbamylase plus ornithine transcarbamylase respectively. That is, uracil prevents functional carbamylphosphate synthetase from being formed by repressing the formation of aspartate transcarbamylase thereby precluding subunit assembly. These studies are directed toward an understanding of the biochemical basis of cold-sensitivity; carbamylphosphate synthetase is a particularly attractive model system because most of the phenotypes of mutations in the genes encoding this enzyme are expressed as cold-conditional. These studies as well as our earlier studies on cold-sensitive ribosomal mutants establish a close correlation between cold-sensitivity and subunit assembly.